The pigment epithelium derived factor (PEDF) is a member of the serine protease inhibitors (serpin) superfamily, but as of today was not found to exhibit inhibitory activity against any proteases. It was first isolated based on its ability to convert dividing retinoblastoma cells into differentiated neurons, and thus was characterized as a neurotrophic factor. Later on it was shown that besides its neurotrophic functions, PEDF is a potent natural inhibitor of angiogenesis in the eye, where it inhibits stimulatory activity of several strong proangiogenic factors. This anti-angiogenic potency has also been shown in several animal models in which PEDF was demonstrated as the factor responsible for the reduction of blood vessel growth in the eye. Although originally discovered in the culture medium of pigment epithelial cells obtained from the fetal human retina, it is clear today that PEDF is expressed not only in the retina, but also at multiple sites in the adult eye, as well as in the adult human brain, the spinal cord, and human plasma. Therefore, it is possible that PEDF has the potential to inhibit angiogenesis throughout the body.
It is well established that protein phosphorylation plays a key role in the regulation of most intracellular processes. However, it is becoming increasingly evident that protein kinases can also regulate extracellular processes, as they are present extracellularly as either ecto- or exo-protein kinases. The ecto-protein kinases are membrane-bound enzymes whose catalytic activities are localized on the extracellular cell surface of a wide variety of cells. The exo-protein kinases are secreted, soluble enzymes whose catalytic activities are present in the extracellular environment without being directly associated to cells. These protein kinases were shown to phosphorylate both extracellular soluble substrates as well as cell surface proteins, thereby playing a regulatory role in many physiological processes including cell-cell interaction, differentiation, proliferation and ion fluxes.
In their recent work the inventors of the present invention have shown that PEDF purified from human plasma (plPEDF) is a phosphoprotein, which is phosphorylated in the circulation by casein kinase CK2 (CK2) and protein kinase A (PKA) (Maik-Rachline G., et al. Blood. 105:670-678, 2005). It was shown that CK2 phosphorylates PEDF on two main residues, Ser24 and Ser114, while PKA was shown to phosphorylate PEDF on Ser227. Using several phosphorylation site mutants that mimic either the phospho (Ser to Glu) or non-phospho (Ser to Ala) forms of PEDF, it was found that each of the CK2 and PKA phosphorylations of PEDF markedly affects its physiological function. The CK2 phosphorylated PEDF had a reduced neurotrophic activity while its anti-angiogenic activity was significantly increased. On the other hand, PKA phosphorylation reduced PEDF anti-angiogenic activity but had very little effect on its neurotrophic activity (Maik-Rachline G., et al. Blood. 2005, 105:670-678).
International Application Publication No. WO 2006/054278 to the applicants of the present invention discloses PEDF variants having substitutions of the two serine residues at the CK2 phosphorylation sites of PEDF, i.e., serine 24 and serine 114, to negatively charged amino acids which exert higher anti-angiogenic activity but lower neurotrophic activity than that of recombinant wild-type PEDF, and PEDF variants having a substitution of the serine residue at the PKA phosphorylation site, i.e., serine 227, to a negatively charged amino acid which exhibit lower anti-angiogenic activity but similar neurotrophic activity to that of recombinant wild-type PEDF. WO 2006/054278 claims anti-angiogenic variants of PEDF comprising at least one altered phosphorylation site. WO 2006/054278 further claims isolated polynucleotides encoding the anti-angiogenic variants of PEDF and methods of use thereof.
U.S. Pat. No. 5,840,686 discloses nucleic acids that encode PEDF, a truncated PEDF, and equivalent proteins, recombinant methods for producing recombinant PEDF, truncated PEDF, and equivalent proteins, and uses of these proteins in neuronal differentiation, neuron survival, and glial inhibition. U.S. Pat. No. 6,319,687 claims a recombinant PEDF protein and truncated forms of PEDF having PEDF biological activity.
International Application Publication No. WO 03/059248 discloses human plasma PEDF that exhibits potent anti-angiogenic and neurotrophic activities.
International Application Publication No. WO 2004/028559 discloses PEDF fragments consisting essentially of 5-50 contiguous amino acids of PEDF, pharmaceutical compositions comprising same and uses thereof for treating cancer or opthalomological diseases.
U.S. Patent Application No. 2005/0222031 discloses a method for preventing or treating malignant melanoma comprising administering a PEDF or a variant of PEDF, which has the functionally equivalent properties to PEDF, to a subject in need thereof. According to U.S. Patent Application No. 2005/0222031, a PEDF variant comprises an amino acid sequence that contains alteration of one or more amino acid residues in the amino acid sequence of human PEDF and has the functionally equivalent properties to human PEDF. However, nowhere in U.S. Patent Application No. 2005/0222031 the position and number of altered amino acid residues are disclosed nor the consequences of amino acid alterations on PEDF biological activity.
U.S. Pat. No. 6,821,775 discloses a replication deficient adenoviral vector comprising a nucleic acid sequence encoding PEDF or a therapeutic fragment thereof. Though suitable for incorporation into the viral vector are nucleic acid sequences comprising substitutions, deletions or additions, which encode a functioning PEDF peptide or therapeutic fragment thereof, U.S. Pat. No. 6,821,775 does not disclose specific PEDF variants capable of exerting superior activity than PEDF.
U.S. Patent Application Publication No. 2003/0158112 discloses a method of treating choroidal neovascularization comprising directly administering a therapeutic factor or a nucleic acid sequence encoding a therapeutic factor to the eye to selectively induce apoptosis of endothelial cells associated with neovascularization of the choroid, such that choroidal neovascularization is treated. Among the therapeutic factors listed is PEDF or a fragment thereof. There is no indication in U.S. Patent Application Publication No. 2003/0158112 for PEDF variants having amino acid substitutions at specific sites, which variants can be used for treating choroidal neovascularization.
International Application Publication No. WO 05/041887 discloses methods for treating conditions involving increased vascular permeability comprising administering PEDF or a PEDF 44 amino acid peptide or a homolog thereof wherein amino acid residues glutamate, isoleucine, leucine and serine at positions 101, 103, 112 and 115, respectively, are unchanged. Nowhere in International Publication Application No. WO 05/041887 it is disclosed that PEDF variants comprising substituted serine residues at positions 24, 114 and 227 can be useful for treating conditions involving increased vascular permeability or increased angiogenesis.
There is still an unmet need for potent anti-angiogenic variants of PEDF that exhibit neurotrophic activity.